MBGE 206 Renaturation Kinetics

Melting temperature

the midpoint of the temperature range for denaturation

Complexity

length in nucleotides of longest non-repeating sequence that can be formed by splicing together all unique sequence in a sample

Rough overview of renaturation kinetics

1-) DNA is sheared to small pieces 2) Heat is used to open up duplex DNA (denaturation) 3)Temperature is lowered to allow DNA to renature (anneal) 4) Reassociation is monitored

What does DNA renaturation rate depend on?

rate depends on concentration and sequence complexity

Relation of repeated sequences with renaturation rate

Fragments containing the more numerous repeated sequences will renature more rapidly than the fragment containing the portions of unique sequences

Formula and its components for renaturation rate

C/C0 = 1/(1+kC0t)

C0t1/2 = 1/k

C = concentration of ssDNA at time t

C0 = initial concentration of ssDNA

t = time in seconds

k = constant

Why doesnt the human genome produce a simple sigmoidal curve when renaturing?

Because it has regions of differing repetitivenes. Purified human DNA does not reanneal as a simple sigmoidal curve, but is composed of the sum of various components.

Significance of C₀t curve

C₀t curve is a measure of sequence complexity

Procedure for Cot analysis

1) Disruption of tissue 2)Phase-coontrast microscopy to check for contaminating organelles and DNA isolation 3) Shearing of DNA 4) Gel electrophoresis to check for fragment size and preparing aliquots of known concnetration 5) DNA solutions are distributed into glass ampuoles each containing same mass of DNA. 6)Tube is placed in boiling water to denature DNA 7) Tube is moved to a water bath in melting temperature. 8)Once desired Cot value is reached, end of tube is broken and sample is diluted with 0.03M SPB. 9)Sample is loaded onto HAP column. 10) After all solution flows through spectrometer and is collected back, the sample is diluted with 0.12 M and loaded onto a new polypropylene column (for elution of ssDNA) 11) 0.5M SPB is added to new column for dsDNA elution 12)Volumes of eluants are determined 13) DNA is denatured with KOH 14)Spectrophotometer readings are taken 15)Graph is plotted